Modied Taohong Siwu Decoction Improved Heart Function After Myocardial Infarction by Inhibiting Inammatory Factor and Promoting Chemotactic and Pro-Angiogenic Factors

Traditional Chinese medicine has been applied to prevent and treat myocardial infarction (MI) in the clinic for a long history. We recently found that Taohong Siwu decoction (THSWD) exerted a benecial effect on heart function after MI through improving the local hostile microenvironment. However, the improvement of cardiac function after THSWD administration was moderate. In this study, four Chinese medicine herbs, which have the properties of warming Yang or removing phlegm, were added into THSWD to constitute a new and multifunctional Chinese herbal compound, named modied THSWD (MTHSWD).


Background
Cardiovascular disease has become the most common cause of morbidity and mortality worldwide, leading to enormous suffering and burden to the families and society. Myocardial infarction (MI), one of the most serious types of cardiovascular disease, is the condition of irreversible necrosis and loss of cardiomyocytes mostly due to myocardial ischemia and hypoxia after the occlusion of coronary artery.
The necrotic cardiomyocytes are gradually replaced by broblasts and collagen tissue, causing the deposition of collagen and formation of brosis scar, resulting in the decline of regional myocardial contractility, even heart failure and death.
Myocardial perfusion with percutaneous coronary intervention (PCI) plays a major role in the prevention of left ventricular remodeling and cardiovascular events in post-MI patients. However, the incidence of heart failure remains high even after the PCI treatment. Although several clinical trials have shown that the bene ts of prevention pharmacological agents, such as angiotensin-converting-enzyme inhibitors/angiotensin II receptor blockers, mineralocorticoid receptor antagonists, beta blockers, aspirin and statins aimed at preventing post-MI remodeling, these interventions remain underutilized in the clinical practice [1]. Emerging data of animal experiments and clinical trials has indicated that stem cell transplantation has potential bene ts in inhibiting left ventricular remodeling and improving heart function after MI [2,3]. According to our previous studies, transplantation of mesenchymal stem cells (MSCs) after MI reduced scar size and improved cardiac function probably via paracrine mechanisms [4,5]. However, its clinical application has been signi cantly hampered by many problems, such as the low survival, differentiation and integration rate after transplantation.
Traditional Chinese medicine (TCM) has a long history in China and has gained multiple clinical applications with multi-target, multi-pathway, low side effects and low cost. In recent years, more and more TCM has been applied to prevent and treat MI in the clinic whether it was used alone or as a complementary treatment [6,7]. Early administration of TCM reduced the rates of in-hospital bleeding and the risk of hyperlipidemia. It was reported that Salvia miltiorrhiza, also known as Danshen, was most commonly used in the post-MI patients [7]. The formulation ShenZhuGuanXin Granules dose-dependently attenuated cardiac dysfunction and enhanced angiogenesis in MI rats through upregulating the expression of PECAM-1/CD31 and vascular endothelial growth factor (VEGF) [8]. A double-blind, randomized, placebo-controlled, pilot study has demonstrated that Danlou tablets signi cantly reversed adverse left ventricular remodeling in patients with MI [9]. However, the sample sizes for most clinical trials were small and the bene cial and detrimental effects of TCM in post-MI patients should be critically evaluated with more studies in the future.
Recently, we found that Taohong Siwu decoction (THSWD) exerted a bene cial effect on heart function after MI through improving the local hostile microenvironment and decreasing the expression of Fis1 [10].
However, the improvement of heart function after THSWD administration was moderate. Further investigations are needed to evaluate if the change in the utilization of Chinese herbal formulations impacts the effectiveness of the treatment. The main effects of THSWD on the patients with MI are "enhancing qi and accelerating the blood circulation" according to TCM theory. Nevertheless, the TCM syndrome typing of MI includes not only "Qi stagnation and blood stasis", but also "Yang de ciency and cold coagulation, Qi stagnation and phlegm obstruction". Therefore, the combination of THSWD with other TCM herbs, which have the properties of warming Yang or removing phlegm, may be more conducive to the improvement of heart function after MI.
Given the above, four TCM herbs including Salvia miltiorrhiza, Radix Astragali,Epimedium and Notopterygii Rhizoma et Radix, were added into THSWD according to the understanding of etiology and pathogenesis of MI in TCM to constitute a new and multifunctional Chinese herbal compound, named modi ed THSWD (MTHSWD). The purpose of this study was to evaluate whether MTHSWD has a better therapeutic effect in a rat MI model. The expressions of cytokines, including chemotactic and in ammatory factors, were examined by ELISA in the infarcted myocardium and serum. In addition, the level of p-Akt and VEGF in the damaged myocardial tissues was further detected to elucidate the possible mechanisms underlying the cardioprotective effects of MTHSWD.

Materials And Methods
Animals Thirty-ve healthy male SD rats (200 ± 20 g) were obtained from the Experiment Animal Center of Shanghai University of Traditional Chinese Medicine. The animals were received humane care and kept on a 12-hours light/dark cycle with free access to water and food. Temperature and relative humidity in the animal breeding room ranged between 23~25℃ and 50~70%, respectively. The protocol was approved by the animal ethics committee of Shanghai University of TCM and the Animal Research Committee of Shanghai. Every effort was made to minimize animal suffering and the number of rats used during experimental manipulation.

Production of MI model
The rats were xed on the surgical plate in a supine position after being anesthetized by 1% sodium pentobarbital (40 mg/kg, i.p.). The rats were orally intubated and mechanically ventilated with a rodent ventilator (Harvard Apparatus, USA). Under sterile conditions, the heart was exposed through a lateral thoracotomy. After that, the pericardium was gently removed and the left anterior descending coronary artery was permanently occluded at 2 to 3 mm below the starting point which is located between the pulmonary arterial cone and the left atrial appendage. Acute MI was con rmed by electrocardiography with ST elevation appearance and visual inspection for a rapid whitish discoloration of anterior wall of the LV. The muscle layer and skin were sutured, and the rats were allowed to recover on a warm heating pad (World Precision Instruments Inc., FL, USA).

Intragastric administration of MTHSWD
There were 5 rats were died during and after the surgery. The survival MI rats were then divided into three groups randomly at the second day after modeling, including MI group, THSWD group and MTHSWD group with 10 rats in each group. The rats in THSWD group or MTHSWD group were intragastrically administered THSWD or MTHSWD respectively twice a day for 2 weeks (1 ml each time). The rats in MI group were intragastrically administered an equivalent amount of physiological saline for the same time. The composition and dosage of THSWD were the same as that used in our published article [10].
MTHSWD for each rat contained 10 components including Semen Persicae (0.16 g), Flos Carthami (0.16 g), Angelica Sinensis (0.22 g), Radix Paeoniae Alba (0.18 g), Rhizoma Chuanxiong (0.14 g), Radix Rehmanniae Praeparata (0.27 g), Salvia miltiorrhiza (0.54 g), Radix Astragali (0.4 g),Epimedium (0.27 g) and Notopterygii Rhizoma et Radix (0.27 g), which were provided by Shuguang Hospital a liated to Shanghai University of Traditional Chinese Medicine. The doses of MTHSWD for rat was converted from a human equivalent dose commonly used in the clinical practice based on the body surface area. THSWD and MTHSWD were prepared according to our reported method [10]. In brief, the crude herbal drugs were mixed and extracted with boiling water. The decoction was concentrated through rotary evaporation under vacuum to an equivalent 2.61 g/mL of the crude herbal drugs.

Echocardiography
Echocardiography was performed on iso urane anesthetized rats with a Vevo 2100 system (Visualsonics, Toronto, Canada) by an operator in a blinded manner at 1 and 2 weeks after treatment. M-mode images of the left ventricle were captured in the parasternal long-axis view and the left ventricle internal diameter at end-diastole (LVIDd) and end-systole (LVIDs) were measured for three consecutive cardiac cycles. The left-ventricular end-systolic (LVESV) and end-diastolic volume (LVEDV) were calculated according to LVIDs and LVIDd, and the ejection fraction (EF) and fractional shortening (FS) of LV were calculated as (LVEDV-LVESV)/LVEDV (%) and (LVIDd-LVIDs)/LVIDd (%), respectively.
Terminal dUTP nick-end labeling assay The heart of rat was rapidly excised under anesthesia after the echocardiographic assessment and was cut into two parts transversally along the center of the infarct zone. The tissue near cardiac base was xed with 4% formaldehyde solution after removing the atrium. Then the sample was embedded in

Statistical Analysis
All data were represented as means ± standard deviation. Multiple groups were compared using one-way analysis of variance (ANOVA) followed by a Student-Newman-Keuls (SNK) post hoc test through a statistical software (IBM SPSS Statistics for Windows, Version 23.0, IBM Corp., NY, USA). Differences were considered to be statistically signi cant when P < 0.05.

Results
MTHSWD improved heart function of MI rats One week after gavage, THSWD and MTHSWD could improve the EF and FS values of MI rats, but the differences between the treatment groups and MI group were not statistically signi cant (Fig. 1A, B). At 2 weeks after treatment, the EF and FS values of MTHSWD group were higher than those of THSWD group, but there was no statistical difference. However, compared with the MI group, the EF and FS values in the MTHSWD group were signi cantly higher (Fig. 1C, D). There were no signi cant differences in the values of LVIDd and LVEDV among the three groups at 2 weeks after treatment (Fig. 1E, G). However, the values of LVIDs and LVESV in the MTHSWD group were signi cantly lower than those in the MI group (Fig. 1F, MTHSWD reduced cell apoptosis in the infarcted area by activating Akt signal TUNEL staining was used to detect the number of apoptotic cells in the infarcted area ( Fig.2A). The number of apoptotic cells in the MI group was the largest, and THSWD and MTHSWD could obviously attenuate the cell apoptosis. Moreover, the number of apoptotic cells in the MTHSWD group was signi cantly lower than that in the THSWD group (Fig.2B). To further investigate the mechanism of cardioprotection by MTHSWD, Western blot was used to detect the level of p-Akt and the expression of cleaved caspase 3. MTHSWD could signi cantly increase the level of p-Akt and reduce the expression of cleaved caspase 3, compared with that in the MI group and THSWD group (Fig.2C-E).

Effects of MTHSWD on the level of cytokines in the infarcted myocardium and serum
The levels of insulin-like growth factor-1 (IGF-1), stromal cell-derived factor-1 (SDF-1), stem cell factor (SCF), vascular cell adhesion molecule-1 (VCAM-1), interleukin-1β (IL-1β) and tumor necrosis factor alpha (TNF-α) in the infarcted myocardium and serum were measured by ELISA. THSWD had no signi cant effects on the levels of these cytokines, but MTHSWD could signi cantly increase the contents of IGF-1, SDF-1 and TNF-α, and signi cantly reduce the expression of IL-1β, in the infarcted myocardium, compared with that in the MI group. MTHSWD did not affect the levels of SCF and VCAM-1 in the infarcted myocardium. There were no signi cant differences in the levels of these cytokines between the MTHSWD group and THSWD group (Fig.3A-F). Similarly, THSWD had no signi cant effect on cytokines in the serum. However, compared with the MI group, MTHSWD could signi cantly increase the expression of IGF-1, SDF-1 and SCF. The levels of VCAM-1, IL-1β and TNF-α in the serum were not affected by MTHSWD. Interestingly enough, the level of SCF in the serum in the MTHSWD group was much higher than that in the THSWD group (Fig.3G-L).
MTHSWD decreased infarct size and collagen deposition in the infarcted area According to the Masson's trichrome staining of heart tissues, the rats in the MI group showed a large area of infarction, while the animals in the MTHSWD group showed the smallest infarct size (Fig.4A). The infarct size in the THSWD group was reduced compared to that in the MI group, but the difference was not statistically signi cant. However, the infarct area in the MTHSWD group was signi cantly lower than that in the MI group and the THSWD group (Fig.4B). In the MI group, the deposition of collagen bers in the infarct area was very obvious and the myocardium was robustly replaced by collagen bers. Intragastric administration of THSWD and MTHSWD could reduce the collagen deposition in the infarct area (Fig.4C). According to statistical analysis, the collagen deposition in the infarction area of the MTHSWD group was the lowest, which was signi cantly lower than that of the MI group and the THSWD group (Fig.4D). Western blot was used to further detect the expression of MMP-2 and TIMP-2 in the infarcted myocardium (Fig.4E). Both THSWD and MTHSWD could down-regulate the expression of MMP-2, and the expression of MMP-2 in the MTHSWD group was signi cantly lower than that in the THSWD group (Fig.4F). In addition, compared with the MI group and THSWD group, administration of MTHSWD could signi cantly up-regulate the expression of TIMP-2 (Fig.4G).
MTHSWD increased the expression of cTnT and Cx43 in the infarcted area Immuno uorescence staining showed that the cardiac-speci c markers cTnT and Cx43 were rarely expressed in the infarcted area of the MI group. The expression of cTnT and Cx43 in the infarcted area of the hearts could be obviously observed in the THSWD group and MTHSWD group (Fig.5A). The relative levels of cTnT and Cx43 in the THSWD group and MTHSWD group were signi cantly higher than those in the MI group. Compared with the THSWD group, the expression of cTnT and Cx43 in the MTHSWD group was signi cantly increased (Fig.5B, C), suggesting that MTHSWD administration played a de nite role in reducing and preventing the loss of cardiomyocytes and electrical coupling between cardiomyocytes.

MTHSWD promoted angiogenesis by increasing the expression of VEGF
The vascular density in the infarcted and peri-infarcted area of rat hearts was determined by the immuno uorescence staining for CD31 and α-SMA (Fig. 6A, B), as the blood perfusion of the lesion was a key factor for the survival of cardiomyocytes and cardiac repair after MI. Both THSWD and MTHSWD could increase the number of microvessels and the number of α-SMA positive vessels in the infarcted and peri-infarcted area. Compared with the THSWD group, the number of microvessels was prominently elevated in the MTHSWD group, but there was no signi cant difference in the number of α-SMA positive vessels between the two groups ( Fig. 6C, D). To further explore the possible mechanism underlying the roles of MTHSWD for the enhanced angiogenesis after MI, the expression of VEGF in the infarcted myocardium tissues was detected by Western blot (Fig. 6E). Compared with the MI group, the expression of VEGF in the THSWD group and MTHSWD group was signi cantly increased. Moreover, the expression of VEGF in the MTHSWD group was higher than that in the THSWD group, suggesting that MTHSWD administration may enhance angiogenesis by increasing the expression of VEGF in the infarcted tissues.

Discussion
MI is the most serious and life-threatening conditions among cardiovascular diseases. Current data suggest that TCM is emerging as a potential treatment for MI in clinical practice. TCM treatment may be bene cial in reducing the mortality of MI patients and improving their quality of life [11]. According to our previous study, THSWD was bene cial for the improvement of heart function after MI. However, this effect was modest although the local hostile microenvironment was improved and mitochondrial ssion was decreased [10]. In this study, THSWD was modi ed by adding four more TCM herbs to constitute a new Chinese medicine prescription, MTHSWD. Interestingly, MTHSWD effectively improved cardiac function, decreased infarct size and collagen deposition in a rat model of MI. More importantly, the infarct size and collagen deposition in the infarcted area were both signi cantly reduced in the rats of MTHSWD group compared with those in the THSWD group. These positive effects, to some extent, may be attribute to the addition of four TCM herbs with the properties of warming Yang and removing phlegm. However, whether these four TCM herbs could exert effects, or even be equivalent to the effects of MTHSWD, deserves further investigation. Besides, the long-term effects of MTHSWD on heart function and its sideeffects should be explored in the future.
It has been reported that the supplemented four herbs have good effects on the patients with cardiovascular diseases. Salvia miltiorrhiza, belongs to the family Labiatae, has a relatively high safety pro le and has been widely used in Asian countries for treating cardiovascular diseases [12]. As a promising candidate for treating cardiovascular diseases, has numerous cardioprotective effects, such as antioxidative, anti-in ammatory, inhibition of apoptosis and anti-cardiac brosis [13]. Salvia miltiorrhiza and Carthamus tinctorius extract has been documented to prevent myocardial brosis and adverse remodeling after MI by epigenetically suppressing Smad3 expression [14]. Radix Astragali is often used in a variety of Chinese herbal preparations as a representative drug for the treatment of Qi de ciency syndrome. Radix Astragali has numerous cardioprotective effects, including reducing in ammatory factor injury and myocardial apoptosis, promoting cardiac microvessel formation and relieving excessive oxidative stress [15][16][17][18]. A recent study showed that the combination of Radix Astragali and Angelica Sinensis not only promoted angiogenesis but also suppressed in ammation and cardiomyocyte apoptosis in a mouse model of MI [19]. The traditional Chinese herb Epimedium has the properties of warming Yang and has been used for the remedy of cardiovascular disease as it has anti-heart failure effect. The ethanol extract of Epimedium ameliorated LV dysfunction, cardiac remodeling and myocardial apoptosis in rats with congestive heart failure [20]. As a major component of Epimedium, Icariin attenuated myocardial infarct size and cell apoptosis by activating the PI3K/Akt/eNOS-dependent signal pathways [21]. In addition, Icariin ameliorated diabetic cardiomyopathy by preventing mitochondrial dysfunction through the Apelin/Sirt3 pathway [22]. Notopterygii Rhizoma et Radix has been widely used in clinical prescriptions to disperse cold and eliminate dampness in China. Modern pharmacological research indicated that Notopterygii Rhizoma et Radix has numerous bene cial effects, including antioxidant, anti-in ammatory and hepatoprotection [23]. Notopterygii Rhizoma et Radix was innovatively used in this study to improve the e cacy in consideration of its effect of dredging collaterals and relieving pain. Although MTHSWD reduced cell apoptosis and improve heart function, it is valuable to clarify the active ingredients of MTHSWD after administration. In addition, it's very interesting to investigate the effects of active ingredients on myocardial damage, thus providing reference data for new drug development.
The loss of cardiomyocytes due to ischemia and hypoxia is the characteristic feature of MI. Indeed, the cell apoptosis and brosis in the infarcted area extensively lead to heart dysfunction. In this study, we found that MTHSWD signi cantly attenuated cell apoptosis probably through activating Akt signaling pathway. PI3K/AKT has been well documented in mediating cell survival, growth, proliferation and differentiation. A recent study indicated that Astragaloside IV inhibited cell apoptosis after MI via regulating PTEN/PI3K/Akt signaling pathway [24]. Meanwhile, we found that the expression of cleaved caspase 3 in the MTHSWD group was much lower than that in the MI group and THSWD group. Caspase-3 is a critical executioner of apoptosis and is responsible for the proteolytic cleavage of many apoptosisrelated proteins. Lentivirus mediated interference of Caspase-3 expression could reduce the infarct size and cell apoptosis as well as improve the heart function in MI rats [25]. In addition, long-term infusion of caspase inhibitor reduced myocardial troponin-I cleavage and preserved myocardial contractile proteins after MI [26]. Interestingly, MTHSWD administration signi cantly increased the level of IGF-1, which is a hormone that promotes growth and prevents cell death. IGF-1 could activate intracellular Akt phosphorylation and decrease caspase activation by binding to its receptor, thus reducing cardiomyocyte apoptosis [27]. Local delivery of IGF-1 by biotinylated nano bers activated Akt, decreased caspase-3 cleavage and improved systolic function after experimental MI [28]. Furthermore, the expression of cTnT and Cx43 in the infarcted area were signi cantly preserved in the hearts of MTHSWD treated rats, suggesting that MTHSWD played a de nite role in preventing and reducing the loss of cardiomyocytes.
The in ammation is elevated and viewed as a major determinant of cardiac remodeling and function after MI. Therefore, the effects of anti-in ammatory drugs were evaluated in patients with MI [29]. MTHSWD could signi cantly reduce the expression of IL-1β in the infarcted myocardium compared with that in the MI group. However, administration of THSWD didn't decrease the levels of IL-1β in the infarcted tissues. As an ancient and evolutionary conserved cytokine, IL-1β is toxic for the cardiomyocytes [30]. The interleukin-1 receptor antagonist inhibited apoptosis and remodeling in experimental acute MI [31]. miR-132 inhibited cardiomyocyte apoptosis and myocardial remodeling after MI through downregulating IL-1β [32]. Interestingly, we found that MTHSWD signi cantly increased the level of TNF-α, which is an important proin ammatory factor, in the infarcted myocardium. It has been proposed that in ammation may have protective or detrimental effects on heart function with the progression of myocardial ischemia [33]. For example, although TNF-α was excessively elevated and contributed to cell apoptosis and cardiac dysfunction after myocardial ischemia-reperfusion, the application of TNF-α inhibitor markedly promoted myocyte apoptosis and reduced cardiac function after MI [34]. Therefore, the exact effect of TNF-α during the progression of myocardial ischemia should be further clari ed through more investigations.
Besides the in ammatory factors, MTHSWD also signi cantly increased the level of SDF-1 in the infarcted myocardium and the levels of SDF-1 and SCF in the serum. SDF-1 is the most paramount chemokine that protects heart from ischaemic injury by recruiting stem/progenitor cells from bone marrow to the site of injury [35]. The interaction of SDF-1 with the receptor CXCR4 and the local concentration of SDF-1 is critical to induce chemotactic response. However, the concentration of SDF-1 in heart is declining by the following days after MI [36]. In addition, the systemic administration of SDF-1 failed to accumulate at the infarcted area. A growing body of evidence suggests that adenoviral delivery of SDF-1α post-infarction effectively improved retention of BM-derived stem-cells, reduced infarct size and improved heart function [37,38]. Accumulating evidence suggests that SDF-1 could also promote angiogenesis after MI [38,39]. The VEGF expression level was obviously increased after the injection of AdV-SDF-1 into the infarcted myocardium [38]. VEGF is well-known for its primary role in inducing and promoting the angiogenesis [40], and MTHSWD obviously enhanced the expression of VEGF in the myocardium tissues. Therefore, the promotion of angiogenesis by MTHSWD may be related to the upregulation of SDF-1 and VEGF after MI. SCF is related to cell migration, proliferation, and survival by binding to the c-kit receptor. It is now accepted that SCF induces mobilization of stem cells from the bone marrow and plays an important role in cardiac repair after MI [41,42]. However, although MTHSWD increased the level of SCF in the serum, the expression of SCF in the infarcted myocardium was not changed obviously after MTHSWD administration.
The stem cell-based therapy is a potential therapeutic strategy for MI, but it was dramatically hampered by the low enrichment in the infarcted tissues after intravenous transplantation. Tanshinone IIA increased the mesenchymal stem cells (MSCs) migration to infarct region via up-regulating SDF-1/CXCR4 signaling pathway [43]. We have observed that Guanxin Danshen formulation promoted the migration and survival of the intravenous-injected MSCs after MI [44]. We proposed that MTHSWD could enhance the engraftment and survival of stem cells in the infarcted area after intravenous injection as it not only raised the expression of SDF-1 and SCF but also reduced the level of in ammatory factor. Therefore, more research is needed to validate effects of MTHSWD combined with the intravenous injection of MSCs or other types of stem cells for the treatment of MI.

Conclusions
The administration of MTHSWD reduced cell apoptosis, promoted angiogenesis and improved heart function after MI. More importantly, the effects of MTHSWD were superior to THSWD in improving heart function and reducing infarct size. This study also indicated that the improvement of cardiac repair after being treated by MTHSWD may be attributed to the downregulation of in ammatory factor IL-1β, upregulation of chemotactic factors SDF-1 and SCF as well as pro-angiogenic factor VEGF, and activation of Akt signaling pathway. Therefore, the novel Chinese medicine MTHSWD proposed in this study has practical signi cance and application potentials for the treatment of cardiovascular diseases such as MI.     Scale bar: 50 μm. (D) Quantitative analysis of collagen deposition in the infarcted area. *P < 0.05 and **P < 0.01versus the MI group. #P < 0.05 and **P < 0.01 versus the THWSD group. n = 10. (E) The expressions of MMP-2 and TIMP-2 in the infarcted myocardium were detected by Western blot. (F, G) The semiquantitative data of Western blots for MMP-2 and TIMP-2. *P < 0.05 and **P < 0.01versus the MI group. #P < 0.05 versus the THWSD group. n = 5.